Synthesis and antifungal activity of chitosan-silver nanocomposite synergize fungicide against Phytophthora capsici -

Synthesis and antifungal activity of chitosan-silver nanocomposite synergize fungicide against Phytophthora capsici

Fungicides are important tools for preventing pathogens and maintaining crop quality; however, their effectiveness was directly affected by high-priced, toxicity, and environmental pollution. In this study, silver-incorporated chitosan nanocomposites (Ag@CS) were first prepared in which CS was used as reducing and stabilizing agent and then these nanocomposites was synergized with fungicide Antracol (An), Ag@CS/An, against Phytophthora capsici causing Phytophthora blight in pepper. The obtained nanocomposites were characterized by Fourier transform infrared (FTIR), X-ray diffraction analysis (XRD), thermogravimetric analysis (TGA), dynamic light scattering (DLS), and transmission electron microscopy (TEM). These results showed that Ag@CS and Ag@CS/An were successfully synthesized with spherical shape AgNPs having diameter of 20.3 ± 0.7 nm and 44.6 ± 0.3 nm, respectively. More importantly, Ag@CS/An was found to have significantly stronger antifungal ability than each component alone, analyzed by agar diffusion method. It might be anticipated that Ag@CS/An has a promising future as nano-antibiotic materials for agriculture.

Synthesis of the AgNPs, Ag@CS and Ag@CS/An

AgNPs were synthesized using the standard protocol. Initially, AgNO₃ solution (1.5 mL, 1 mM) was mixed with TSC solution (10 mL, 0.25 mM). The ice-cooled NaBH4 solution (20 mL, 10 mM) was immediately added into the above mixture to form AgNPs. The colloected AgNPs were stored in complete darkness to prevent light-induced ion release. Ag@CS was prepared by using AgNO₃ and CS as a reducing and stabilizing agent. Briefly, CS (0.2 g) was dissolved in 1% CH3COOH (10 mL) at pH 3.5, followed by mixing with 2.5 mL of AgNO₃ under constant stirring for 30–45 min. Then, NaOH solution (1 M, 10 mL) was dropped into the obtained AgNO₃-CS solution using syringe pump. After 15–20 min, the collected Ag@CS spheres with yellow-brown color were washed to discard the residues and then stored in a refrigerator for preventing Ag reduction. Lastly, Antracol (An)  dissolved in distilled water was added drop-wised into Ag@CS acidic solution (pH 3.5), followed by constant stirring of the mixture for 24 h at room temperature. Antracol (An) enters the polymeric nanocomposite matrix and finally is wrapped inside the Ag@CS nanocomposite. The volume ratio 2:1 between Antracol (An) and Ag@CS solution was used in the synergistic process.

Anti-fungal effect test of Phytopthora

Antifungal activity was estimated by the agar diffusion method. First, the fungal Phytophthora capsici suspensions were spread on PDA agar plate (potatoes infusion from 200.00 g/L, dextrose 20.00 g/L, agar 15.00 g/L). Next, paper disks (5 nm in diameter) containing Antracol (An),  AgNPs, CS, Ag@CS and Ag@CS/An at different concentrations were placed on each plate and incubated at room temperature for 2-3 days. The Antracol (An) cultured in agar-based growth medium were used as a control and were assigned to 100% survival. The inhibition zone was calculated in diameter. The data were expressed as mean ± SD.

Characterization

The morphological characteristics of the synthesized NPs were determined by TEM using FEI Tecnai G2 20 S-Twin at 100 kV. The hydrodynamic diameter of Ag@CS and Ag@CS/An was measured at 37 °C using a Zetasizer Nano ZS (ZEN 3600, Malvern Instruments, UK), equipped with a 633 nm wavelength HeNe laser, and the scattered light was detected at 90. The sample was sonicated for 10 min, filtered (pore size = 0.45 mm), and loaded into the quartz curvet for measurement. Zetasizer Nano Series Ver. 5.03 software was used for data acquisition. In order to characterize the thermal decomposition profiles of both CS and Ag@CS composites, TGA was performed on a TGA (Q500 V20 Bluid 36) and 5 mg specimens of CS and Ag@CS composites were measured with a nitrogen flow rate at a heating rate of 10 C/m 1 from 50  to 800 °C. The FTIR spectra were recorded with a Spectrum Tensor27 FTIR Spectrometer, using KBr pellets FTIR Spetrometer in the range of 400 to 4000 cm 1 with a resolution of 4 cm1. X-ray diffraction patterns were obtained using a Rigaku D/Max-2550 V diffractometer with 2 V. T. LE ET AL. Cu K-a radiation (k = 1.5406 Å) at a scanning rate of 0.03 s 1 in the 2h range from 10 to 90.

Results

The demand for polymeric layer surrounding AgNPs, in which AgNPs were finely dispersed within polymeric matrix or coated with polymer to form core-shell structure. In this study, chitosan was utilized as a polymeric matrix for AgNPs. In addition to desirable properties including homogenous dispersion and chain structure that enables the incorporation of AgNPs, chitosan is known as a reducing and stabilizing agent for green synthesis of AgNPs.  Silver ions are being coordinated by amino groups of polymeric chains in chitosan acidic solution. Irons reduction to metallic AgNPs is coupled with oxidation of hydroxyl groups of chitosan. As a result, the polymeric nanocomposite system of polymeric chitosan network with embedded AgNPs was created. Besides, chitosan also improves the stability of embedded AgNPs since the connection between chitosan polymeric matrix and AgNPs reduces the tendency of aggregation of AgNPs.

Particles size and morphology

In aqueous solution, the size of Ag@CS was 20.3 ± 0.7 nm. After mixing with An, the particle size of Ag@CS/An was significantly increased to 44.6 ± 0.3 nm. The increase in particle size of Ag@CS/An NPs could be attributed to the loose attachment of An on the surface of AgNPs embedded within Ag@CS matrix. In addition, TEM was used to characterize the morphology and size of Ag@CS and Ag@CS/An. The particle size distribution of Ag@CS and Ag@CS/ An was found to be 12-32 nm and 10-30 nm in diameter, respectively. Both prepared NPs in nanocomposite were spherical in shape, well separated, and reasonably dispersed. The synthesized AgNPs completely separated from each other without clumping, meaning that CS may serve as a stabilizing agent to prevent aggregation of AgNPs and help to narrow size distribution. The obtained Ag@CS and Ag@CS/ An have high permeability for efficient antimicrobial applications.

Anti-fungal activity

Anti-fungal activities were significant differences among using AgNPs, CS, Ag@CS and Ag@CS/An on growth inhibition of Phytophthora capsici. The average diameter of inhibition zones of Ag and CS at concentration of 2 ppm and 4000 ppm were found to be only 5.35 ± 0.58 mm and 6.93 ± 0.78 mm, respectively. When putting them together, the average diameter had approximately doubled to 11.67 ± 0.58 mm. More importantly, when Ag (2 ppm) and CS (4000 ppm) combined with An, the zones of inhibition even more significantly increased, as compared with Ag@CS alone. In detail, although the 500 ppm of An concentration combined with Ag@CS had just about the same inhibition zone as free An (around 11 mm), the combining of 1000, 2500, and 5000 ppm of An with Ag@CS showed large inhibition zones of 19.07 ± 0.15, 23.6 ± 0.7, and 26.44 ± 0.5 mm in diameter, which were dramatical high compared to either Ag@CS or free An at similar concentrations (inhibition zones of 6.30 ± 0.6, 8.10 ± 0.56, 8.60 ± 0.26 mm at 1000, 2500, and 5000 ppm, respectively). It was clear that the antifungal activity of Ag@CS/An was not only proportional to the concentration of An but also synergistic after functionalizing of Ag@CS with An. This will reduce the required concentration for each component while still exhibit high antifungal activity, thus reducing the risk of environmental pollution. These results suggested that Ag@CS/An may serve as antifungal candidate (cost saving, safety and high antifungal effectiveness) against Phytophthora capsici.

Conclusion

In this study, Ag@CS were successfully prepared with An against Phytophthora capsici. The prepared AgNPs in Ag@CS and Ag@CS/An nanocomposite existed in spherical shape with particle size of 20.3 ± 0.7 nm and 44.6 ± 0.3 nm and act as potential material for effective cellular penetration and fungal killing. Especially, the Ag@CS/An showed a highly improved antifungal activity in comparison with each individual component. These Ag@CS/An could be a promising candidate for the next generation of safe and effective antifungal agent.

Citation:

Le, V.T., Bach, L.G., Pham, T.T., Le, N.T.T., Ngoc, U.T.P., Tran, D.H.N. and Nguyen, D.H., 2019. Synthesis and antifungal activity of chitosan-silver nanocomposite synergize fungicide against Phytophthora capsici. Journal of Macromolecular Science, Part A, 56(6), pp.522-528.